Project Summary/Abstract Environmental DNA damaging agents including natural radioisotopes in the soil, UV radiation from the sun, and chemical byproducts present in exhaust fumes constantly threaten the genomic integrity of a cell. Humans are regularly exposed to these environmental factors, and such exposure leads to various forms of DNA damage. When classical DNA repair mechanisms fail, regions of damaged DNA will persist in the genome. These lesions pose a major problem for replication machinery. The replication fork will stall at sites of DNA damage, putting the cell at risk for chromosomal rearrangements and double stranded breaks that often result in cell death. To prevent such catastrophic outcomes, the cell utilizes damage-bypass pathways that allow for continued DNA replication through regions of damage. This research focuses on one such pathway, error-free template switching. Error-free template switching relies upon the activity of the Rad5 protein. Unlike other damage- tolerance pathways, Rad5 rearranges DNA in such a way that regions of damaged DNA are not used as a template during the replication process. As a result, while other damage-bypass pathways inaccurately add nucleotides across from sites of DNA damage (thus introducing disease-causing mutations), error-free template switching always leads to the incorporation of the intended nucleotide, thereby preventing the introduction of mutations. The goal of this research is to understand the mechanism of Rad5 mediated DNA rearrangement in error-free template switching from biochemical and structural perspectives. First, the structure of the Rad5- DNA complex will be investigated. X-ray crystallography and small angle X-ray scattering (SAXS) will be used to study the structure of Rad5. The placement of Rad5 on DNA substrates will be investigated through a nuclease protection footprinting assay. Next, the rate of DNA unwinding, the step size, and processivity of Rad5 will be determined through kinetics assays. Finally, error-free template switching will be reconstituted in vitro, allowing the isolation of a key intermediate in this pathway. Overall, Rad5's role in error-free template switching will be clarified, providing insight into the way cells cope with harmful environmental agents.